SOP 1-8 - PDSP - NIMH Psychoactive Drug Screening...

[18F]FALLYPRIDE FOR INJECTION: STANDARD OPERATING PROCEDURES

PET Radiopharmaceutical Sciences Section, Date of review: 05/17/04Molecular Imaging Branch, National Institute of Mental Health, National Institutes of Health, Bldg. 10, Rm. B3 C338,Bethesda, MD 20892

List of SOPs for [18f]Fallypride for Injection

Name of SOP SOP #

1 Preparation of Stock Solution of Kryptofix 2.2.2* and

Potassium Carbonate

SOP#GP101

2 Cleaning Procedure for Radiochemistry Glassware, Reactor

Vials and Magnetic Bars

SOP # GP102

3 Fallypride HPLC Mobile Phase Preparation SOP # GP103

4 Micropore Filter Testing and Drug Product /Filter

Compatibility (Bubble Point Test).

SOP # GP104

5 Preparation of Stock Acetate Buffer SOP # GP105

6 BOXCLEAN Program (GE Tracerlab FXFN) SOP # MP201

7 GE Tracerlab FXFN Module. Vacuum Pressure Check SOP # MP202

8 Production of [18F]Fallypride for Injection; Part 1: Pre-

Synthesis Procedures

SOP # MP203

9 Production of [18F]Fallypride for Injection; Part 2: Synthesis

and Purification

SOP # MP204

10 Production of [18F]Fallypride for Injection; Part 3:

Formulation

SOP # MP205

11 Standard Curve of Reference Fallypride SOP # QA301

12 Analysis of Organic Residues by Gas Chromatography

in [18F]Fallypride for Injection

SOP # QA304

13 Analytical HPLC QC-Method SOP # QA305

14 Sterility Test SOP # QA306

15 P Pyrogen Test (LAL Bacterial Endotoxin Test).

Si Simplified Procedure

SOP # QA307

16 Analysis of The Purity of Fallypride and Tosyl-Fallypride by

HP HPLC

SOP # QA308

Document 5. [18f]Fallypride for Injection: Standard Operating Procedures of 34



SOP # GP101 Preparation of Stock Solution of Kryptofix 2.2.2* and Potassium Carbonate

Approved by: _____________________________Initials________ Date:___________

Victor W Pike, Ph.D., Chief, PET Radiopharmaceutical Sciences, NIMH

1. Procedure:

1. Using an analytical balance and a plastic screw-cap vial (20 mL size), weight out 25 mg of

potassium carbonate (Aldrich, anhydrous, 99.99%). Add deionized water (0.5 mL) and mix

to dissolve.

2. Using weighing paper, weigh 250 mg of Kryptofix 2.2.2 (anhydrous; 98%; Aldrich) and

transfer to the same vial. Add dry acetonitrile (4.5 mL) and mix to dissolve.

3. Place appropriate label showing preparation and expiry date (to be determined) and keep

the solution in the refrigerator until use (do not store in glass vial).

4. Record the data of Kryptofix 2.2.2-potassium carbonate complex in Table 1.

2. Record: Lot #__________________________

Table 1. Reagents : [18F]fluoride-Kryptofix 2.2.2-potassium carbonate complex

Item Reagent Specifications Manufacturer Lot # Expiry date Quantity

1 Kryptofix 2.2.2

(250 mg)

Anhydrous

98%

Aldrich Chem.

Co.

2 Potassium

carbonate

(25 mg)

Anhydrous

99.99%

Aldrich Chem.

Co.

3 Acetonitrile

(4.5 mL)

Anhydrous 99.8% Aldrich Chem.

Co.

4 Water

(0.5 mL)

De-ionized M; Millipore, In

house

Note: 100 µL of solution contains 0.5 mg potassium carbonate and 5 mg Kryptofix 2.2.2

Chemist_____________________________Initials_________________Date________________


Purpose: to prepare a stock solution of Kryptofix 2.2.2* and potassium carbonate



SOP # GP102Cleaning Procedure for Radiochemistry Glassware, Reactor Vials and Magnetic Bars

Approved by: _____________________________Initials________ Date:_____________

Victor W Pike, Ph.D., Chief, PET Radiopharmaceutical Sciences, NIMH,

Procedure:

1. Rinse with acetone (HPLC grade) to remove organic residues.

2. Rinse with hexane (HPLC grade) to remove residual silicon oil.

3. Bathe the glassware in an aqueous 2% solution of Liqui-Nox (Valconox, made from dilution

of a 10% stock solution).

a) Bring solution to vigorous boiling for 5–10 min.

b) Swab material being washed with wood, cotton-tipped applicators (6 in).

4. Rinse glassware 3 times with Millipore water.

a) Carefully spot-check each item.

b) Give final rinses with de-ionized water bottle (3 times).

c) Leave the clean material to drip, over paper towel.

5. Place small V-vials, magnetic bars and reaction vials in beakers (Pyrex) and cover with

aluminum foil. Place material in the oven at 170−180 ºC and allow to dry overnight.

6. Keep all the material in the oven until use.

Notes:

a) Except for the aqueous Valconox that can be disposed in the sink, all other washing

solvents must be placed in their respective waste container.

b) In the Oven Log enter date and temperature IN and date and temperature OUT.



Purpose: to clean glassware, reactor, vials and magnetic bars that are used in the production of [18F]Fallypride for Injection.



SOP # GP103Fallypride HPLC Mobile Phase Preparation

Approved by: _____________________________Initials________ Date:_____________

Victor W Pike, Ph.D., Chief, PET Radiopharmaceutical Sciences, NIMH,

Procedure:

1. Preparation of HPLC solvent for the preparative column

a) Fill up a flask (1 L size) for HPLC solvent with acetonitrile (HPLC grade) containing

0.6% TEA (6 mL of TEA /L).

b) In a similar flask, prepare 1 L of HPLC water only.

2. Preparation of HPLC solvent for analytical column.

a) To prepare stock ammonium formate solution (100 mM), weigh out ammonium

formate (6.013 g) on weighing paper and then dissolve in HPLC water (1 L), mix well

and store at room temperature.

b) Put stock ammonium formate solution (50 mL; 100 mM) into a volumetric cylinder (1

L size), then dilute with HPLC water (450 mL; HPLC Grade)

c) Put acetonitrile (HPLC grade; 500 mL) into another volumetric cylinder. Mix

solutions from (a) and (b).



Purpose: to prepare mobile phase for the HPLC purification of [18F]fallypride and QC

analysis of [18F]Fallypride for Injection.



SOP # GP104Micropore Filter Testing and Drug Product /Filter Compatibility

(Bubble Point Test).

Approved by: _____________________________Initials________ Date:____________

Victor W Pike, Ph.D., Chief, PET Radiopharmaceutical Sciences, NIMH

Filter-Integrity test. The integrity of the Millex-MP sterile filter that was used for sterile

filtration of [18F]Fallypride for Injection is tested by the following method. The knob on the

pressure regulator that is hooked to the house compressed air gas supply is turned counter

clockwise so as to minimize outlet pressure. The filter assembly is removed from the

sterile dose vial and placed on the male luer-lock fitting of the pressure-regulated 1/8”

Teflon line. A disposable needle (1.5 inch 22 gauge) is placed on the male luer fitting of

the Millex-MP filter. A portion of this needle is submerged in a test tube (12 x 50 mm)

containing about 3 mL of HPLC grade water. The knob of the pressure regulator is slowly

turned clockwise and the pressure gauge is monitored visually. The pressure is brought to

45 p.s.i. If there is an absence of a steady stream of bubbles in the test tube when the

pressure gauge reads 45 p.s.i., then the filter passes the test. The result of the filter test is

recorded on the batch record and in the summary section of the quality control test form.



Purpose: Testing of Micropore filter compatibility.



SOP # GP105Preparation of Stock Acetate Buffer

Approved by: _____________________________Initials________ Date:______________

Victor W Pike, Ph.D., Chief, PET Radiopharmaceutical Sciences. NIMH

A. Reagents:Name of reagent Manufacturer Quantity

Sodium acetate Aldrich Chem. Co.

Cat. # 241245, ACS reagent

Acetic acid Aldrich Chem. Co.

Cat. # A6283, 500 mL

Water EMD Chemicals Inc.

Cat. # WX0004-1

B. Procedure:

1. On weighing paper, weigh out sodium acetate (14.45 g) and then dissolve this in HPLC

water (500 mL) in a bottle (500 mL size).

2. Take acetic acid (2.15 mL) and put it into above solution.

3. Mix well.

4. Measured the pH of the buffer solution; the pH of this buffer should be 5.3−5.7.



Purpose: to prepare stock acetate buffer (250 mM; pH 5.5) for use in [18F]Fallypride for Injection prouction



SOP # MP201BOXCLEAN program (GE TRACERlab FXFN)

Approved by: _____________________________Initials________ Date: _____________

Victor W Pike, Ph.D., Chief, NIMH, PET Radiopharmaceutical Sciences

1. Procedure:

1. If present, remove used Sep-Pak and re-connect line to its holder-connector.

2. Disconnect the following three lines placing the output into the waste reservoir:

a) The one coming from the Sep-Pak holder-connector into waste.

b) The one coming from the Sep-Pak holder-connector, going into the collection V-

vial (10 mL size)

c) The one going from the HPLC loop waste into a V-vial (5 mL size) connected to

an external empty syringe (20 mL size), to pull diluted reaction mixture into

injector loop.

d) A fourth line, from the injector to a Blue Max (50 mL size) or to a vial (20 mL

size) (waste) remains the same.

3. Check that nitrogen and air compressed air valves are open. Check nitrogen cylinder is

open. Be sure there is a reactor vessel connected.

4. Place dry-ice in the vacuum trap.

5. Start the TracerLab automated software program.

Select Synthesis: "Boxclean", ENTER.

Follow instructions.

Add acetone as follows:

a) 1−2 mL acetone to target vial

b) 1–2 mL acetone to support vials V1 to V9 (fill small ones and half fill large ones)

c) 25 mL acetone to collection bulb vessel

d) 0.5 mL acetone to V-vial (10 mL size) (leave the spinal needle way up)


Purpose: to clean the GE TracerLab FXFN module, check its functions and prepare it for production.



6. In Preparation CHECK LIST, check each item in the list, going down with repeated

ENTER. Finally, enter operator initials.

7. The screen will show the list of timed events for BOXCLEAN and the process will start

with the vacuum pup ON and the temperature increasing to 100oC.

Check that the temperature reaches 100oC and the pressure in reactor goes down.

8. At the end of the Program, the system will be flushed with nitrogen, go under vacuum to

remove most of the acetone vapors and then stop.

9. Remove the acetone that may be trapped in the vacuum trap. Dispose of it in the

appropriate container.

Copy of the BOXCLEAN Program see Attached file: Document 9: Validation Runs, attached

BOX Clean Program on the batch: FAY-032404.





SOP # MP202GE TRACERlab FXFN Module. Vacuum Pressure check



Procedure:

1. Open nitrogen and compressed air valves located outside hot-cell

2. Start Dell Computer-Windows 98. Enter Name and Password. After Virus Scan

Computer will open. Click twice on TRACERLab icon. Enter "Admin" and "Tracerlab"

3. Once on the Instrument SCHEME, look at the square area in the right side of the screen

that shows pressure of compressed air and nitrogen.

4. Turn on the vacuum on by click “ON” of the "Power”, which represents the vacuum on

the Instrument SCHEME. (Note: this can be turned OFF in the same way).

5. Compressed air pressure should be around _____kPa and nitrogen around______ kPa.

6. Click the mouse in V24 and V25 should open with space bar (1 = open, 0 = close), this

will set full vacuum on reactor vessel, reaching around ≤ 3 kPa. Record the lowest

pressure. _______kPa

7. Close V24 (space bar), pressure should hold (change should be ≤ 1 kPa/min).

8. If pressure is not holding find and correct the source of leak; e.g. tighten stoppers in

support vials, check valves 2 to 6, 13, 14, 20, 24; tight reactor vial, check O-ring.

9. With mouse, go to V20 (Nitrogen), open V20 with space bar (can also close with space

bar). Pressure should go up. Close with space bar and open V24. Check the pressure

again. It should go below 2 kPa again.

10. If pressure holds, go ahead with the next step.

Note. When both vacuum and pressure are open during the procedure, pressure should hold

between 21.5 to 22 kPa (V20 and V24 open).



Purpose: to check the vacuum efficiency and the leak of GE TRACERlab FXFN module to ensure the success of production synthesis.



SOP # MP203Production of [18F]Fallypride for Injection; Part 1: Pre-synthesis Procedures



Procedures:

1. Check that nitrogen and compressed air valves are open.

2. Check nitrogen cylinder is open.

3. Turn on the computer with TRACERlab FXFN software and with Beckman HPLC

software.

4. Open the TRACERlab FXFN software and check the pressure of compressed air and

nitrogen pressure. The pressure of compressed air should be 400 p.s.i and nitrogen

pressure > 100 p.s.i.

5. Clean GE TRACERlab FXFN Module and hot-cell 5.

4.1 Follow the program "CLEAN BOX" (SOP # MP201). Clean and dry the GE

TRACERlab FXFN module on the day of preparation or the day before the preparation.

All the reagent vessels and transfer lines of the module to be used in the preparation

should be washed with acetone and dried under nitrogen flush before using.

4.2 Inspect hot-cell compounding/dispensing area for cleanliness. Remove extraneous

materials and labels. Spray ethanol to clean this area if necessary.

5.3 Inspect and set if necessary charcoal trap in the module

6. Prepare [18F]fluoride ion-Kryptofix 2.2.2-potassium carbonate complex solution

5.1 Add stock Kryptofix 2.2.2-potassium carbonate (70−100 µL) into V-Vial (1 mL size).

5.2 Take V-Vial 1 to Cyclotron Facility (CC, NIH) to collect aqueous [18F]fluoride ion;

the activity of [18F]fluoride ion should be between 150−250 mCi water ( ≤ 500 µL).


Purpose: to prepare for the synthesis of [18F]Fallypride for injection on the GE TRACERlab FXFN module in hot-cell 5.



6. Verify integrity of glassy reaction vessel assembly (containing small magnetic bar stirrer) in

TRACERlab FXFN Module. Follow SOP # MP202 and record the data in the sheet of

Summary Records of [18F]Fallypride for Injection run:

6.1 Vacuum pressure check (gauge, kPa).

6.2 The compress air pressure (kPa)

6.3 Nitrogen pressure (kPa)

7. Prepare the preparative HPLC system

7.1 Start the HPLC computer software, enter the necessary information and select the

right method “ Fally_Production”

7.2 Verify that mobile phase bottles in the right position on the Beckman System Gold

Delivery module.

7.2.1 The solvent bottle of 0.6% TEA connects to B1 line (marked red)

7.2.2 The solvent bottle of HPLC water connects to A1 line (marked red)

Above connection should match the setup in the method

“Fally_Production”.

7.2.3 Set Acetonitrile w 0.6%TEA /HPLC water (30: 70 v/v) as initial

conditions. Equilibrate the Luna column (Phenomenex) with

fallypride preparative mobile phase (~ 200 mL)

7.3 Note the pump pressure while increasing the flow rate to 2 mL/min.

7.4 Pump the HPLC mobile phase through the preparative column at a low flow (1−2

mL/min) and maintain this flow until purification time.

8. Prepare in the clean bench a sterile filtration/collection Unit, using aseptic technique:

8.1.Prepare the final sterile product vial. The final sterile empty vial, product needle,

vent needle and two filters (Millex 0.22 µm) are assembled in a certified laminar

flow sterile cabinet. Attach a prepared label for [18F]fallypride with the current batch

number and date to the product vial.

8.2.Move a prepared product vial unit from the laminar flow sterile cabinet and attach it

to the product line of TracerLab FN Module.

9. Open the Tracerlab Fx Computer Controlling System ("Admin" and "Tracerlab") and Select

Synthesis method: "Fallypride". Click “Synthesis-Start” to start preparation of the

synthesis.

10. Enter all the required information (including date, radiotracer, batch #, human preparation

#) into the corresponding method of the HPLC computer software program.




11. Check that you have the correct UV absorbance (254 nm) and the correct settings for the

gamma 12. Place the solvents and precursor solution in the corresponding module support

vials:

________Support vial 1: acetonitrile (0.5 mL).

________Support vial 2 [Used as a port (V-vial; 1 mL size)]: [18F]fluoride-K 2.2.2-

potassium carbonate solution; 100 µL).

________Support vial 4: dry acetonitrile (1 mL) plus tosyl fallypride (2.0 ± 0.5 mg).

________Support vial 5: dry acetonitrile (0.5 mL)

________Support vial 6: dry acetonitrile (1.5 mL)

________Support vial 7: sterile saline solution (9–14 mL)

________Support vial 8: Ethyl alcohol for Injection (1 mL)

________Support vial 9 sterile water (8 mL)

_______ Large (10 mL size) V-vial: 0.5 mL sterile water with 0.6%TEA.

_______ Large bulb vessel: HPLC water (90 mL) and sodium acetate (250 mM; pH

5.5; 10 mL; see SOP # GP104).

12. Check that ‘complex solution’ delivery line goes through valve V2 and into reactor vessel

(glassy carbon).

13. Place acetonitrile (0.5 mL) in a syringe (3 mL) connected to an external line going into the

"complex" vial for recovery of [18F]fluoride-K 2.2.2-potassium carbonate solution left in

transportation vial and its addition to the reaction vessel.

14. Activate one C-18 plus Sep-Pak with ethanol (10 mL) sterile water (10 mL) and place in its

corresponding connection site in the module.

15. Place the small double-neck round bottom flask to receive the ethanolic eluate of the Sep-

Pak in its corresponding location in the Box.

16. In a lead pot, lace a sterile empty vial (30 mL size) fitted with a vent needle/filter and a

second needle and Millev GV filter for sterilization of the final product.

17. Place enough dry-ice/acetone mixture in the vacuum trap.

18. Check that the nitrogen and compressed air valves close to the hot cell are open (also

check the nitrogen cylinder).

19. Assure that syringes and needles have been changed. Use depyrogenated reaction vessel

(with magnetic stirrer), HPLC collection bulb (10 mL size) V-vial, two neck round-bottom

flask, sterile vial etc.

20. Keep the HPLC column flow at 2 mL/min.




Synthesis comments:

_______________________________________________________________________________

_______________________________________________________________________________

____________________________________________________________________________

The approved procedure has been followed without any deviation





SOP # MP204

Production of [18F]Fallypride for Injection; Part 2: synthesis and purification



Procedures: [18F]Fallypride synthesis and purification

1. Preparation of [18F]fluoride ion/Kryptofix 2.2.2-potassium carbonate complex ([18O]water-

acetonitrile-Kryptofix 2.2.2-[18F]fluoride ion-potassium carbonate).

1.1.Using the dose calibrator (Biodex AtomLab 300) set at F-18, determine the amount

of radioactivity that will be transferred to the clean glassy reaction vessel (20 mL

size). Record the data: EOB, starting activity of [18F]fluoride-[18O]water and

Volume of [18F]fluoride-[18O]water.

1.2.Connect one vent needle, one needle and line from external syringe containing

acetonitrile (0.5 mL) and one spinal needle and line going from the radioactive

solution through V2 to reactor vial. Make sure that spinal needle is all the way

down to the bottom of the radioactive solution.

2. Starting the preparation and transferring the F-18 complex.

2.1 Start the TRACERLab automated software program. Select Synthesis: "Fallypride"

in method box, click “Synthesis-Start” in main menu bar, follows process

instructions. Organize "Instrument screen", "Program" and "Graphics" in the

computer screen.

2.2 The program will have already started setting the vacuum pump ON and

transferring the F-18 complex from the V-vial (1 mL size) into the reaction vessel,

then MESSAGE BOX comes out. At this moment add acetonitrile (0.5 mL) from the

outside syringe into the transport F-18 vial (pushing with air until the line is empty

and the vial is full). Press “Enter” or click “OK” when done.

3. [18F]Fallypride synthesis step 1: remove water from the F-18 complex.


Purpose: to synthesize and purify [18F]fallypride



The reactor temperature will be increased first to 65 ºC and later to 88 ºC (T1 + 3 min) and

then back to 60 ºC. At this point, the contents of V5 are added and the temperature cycle

starts again: heat at 65ºC, then at 88ºC and finally at 60 ºC (drying process).

4. [18F]Fallypride synthesis step 2: 18F-Fallypride reaction. Check V4 and reaction

temperature.

4.1 Once drying is complete, V4 will open and the tosylate precursor in acetonitrile (1 mL)

will be added to the reaction vessel and the mixture heated at 100 ºC for 30 min. At

the end of the reaction, the reaction vessel will be cooled at 35 ºC and the first part of

the Program will finish.

4.2 A message will shown saying "End of synthesis, Hit OK to continue to next Program".

Hit CONTINUE.

5. Prepare Beckman HPLC system for purification.5.1 Verify that the HPLC is in Preparative and in LOAD and that the detectors are in the

right settings.

5.2 Check that method setting of Beckman HPLC instruments is right

5.3 Set the flow rate to 9 mL/min. Wait for pressure to go up.

5.4 Click “Single run” in Beckman HPLC software to start acquisition and wait the

acquisition Box appears.

5.5 Check that the injector turn to inject position

6. [18F]Fallypride purification step 1: transferring the initial reaction mixture.

6.1 Second program continues purification process. The reaction mixture will be

transfered to the V- vial (10 ml size) containing sterile water (0.5 mL) for pre-dilution

before HPLC injection.

6.2 Check that the injector transfer needle in V- vial (10 mL size) is far up from the level of

the solution.

6.3 Check that valve 6 will open and the acetonitrile (1.5 mL) is added to the reaction

vessel.

7. [18F]Fallypride purification step 2: loading and injection

7.1 After transferring, a message box will appear "System now ready to load Loop. Hit OK

when completed". System will load the reaction mixture solution from T-vial (10 mL

size) into HPLC loop automatically.

7.2 At this moment, new message box will appear “System now ready to inject. Hit OK

when the loading is completed.”




7.3 Watch the 10 mL T-vial closely. While the solution loading completed, hit OK.

7.4 At same time hit “OK to start the HPLC run in Beckman software

8. [18F]Fallypride step 3: HPLC separation and product collection.

8.1 In GRAPHICS observe the UV and and radioactivity traces on the data acquisition

systems.

8.2 When the desired peak start coming out click on the collect arrow. Record the Rt

=____________min.

8.3 The mobile phase containing the product will be added to the bulb vessel containing

sterile water (90 mL) for dilution and of sodium acetate (10 mL) for dilution and

neutralization.

8.4 At the end of collection click on the end collection arrow to stop fraction collection (the

mobile phase will go into the waste reservoir).

Synthesis comments:_______________________________________________________________________________

_______________________________________________________________________________

____________________________________________________________________________






SOP # MP205

Production of [18F]Fallypride for Injection, Part 3: formulation



Procedures: [18F]Fallypride formulation

1. The program will continue Fallypride Production Part 2 and the diluted fraction will be

passed through the solid phase cartridge (SPE; Waters C-18 Sep-Pak) for concentration.

This process is programmed for 6.5 min. After passing through Sep-Pak, valve V9 will open

and Sep-Pak will be washed with sterilized water.

2. After the water wash, V8 will open and the product will be eluted from the Sep-Pak with

ethanol (1mL) into the two-neck round bottom flask.

3. Program will stop to allow sampling of ethanolic solution if necessary. The message "Take

QC sample and hit OK to proceed with filtration" will appear. Take 40-100 µL of sample for

QC from the two-neck round bottom flask if needed.

4. The program can be continued hitting OK. Valve 7 will open and saline solution will be

added to the neck round bottom flask containing [18F]fallypride product.

5. The program can be continued hitting OK. The saline solution will be then transferred to

the sterile and non-pyrogenic vial (10 mL size, Flip-top- Vial – Glass) through the Millex

filter Unit (0.22 µm) for filtration and sterilization.

6. After filtering product, remove sterile filter, measure the activity of the final [18F]fallypride

product, record the number on the master batch Sheet and fill the number in the labels.

Activity of final [18F]Fallypride for Injection for _____________mCi at____________;

The volume:_____mL and the concentration:___________ mCi/mL.

The final Collection Vial and the lead shielded pot will be labeled as follows (21

CFR 361.1):


S Purpose: Formulation of [18F]Fallypride for Injection



[18F]Fallypride for InjectionSterile, apyrogenic solution for intravenous administrationCaution: New drug limited by Federal law to investigational use 0nly

NIMH MIB Store at room temperature Expires 4 h after calibration Activity:______________ mCi Time: _________________ Volume: ___________ mL. Concentration: ______________ Batch #: FAY-________________ Date:_____________

In addition, one of the labels will be attached to the Radiopharmacy Form as record.

7. Sampling for QC: The 1 cc sterile polypropylene syringe may be used to withdraw a 1 mL

sample of the formulated [18F]Fallypride for Injection in a certified laminar flow sterile

cabinet. The 1 mL sample of [18F]Fallypride for Injection is transferred to sterilized

depyrogenated tube labeled “[18F]Fallypride for Injection” and the vial is dated and stored in

a lead pig. This aliquot will be used for the following QC tests: radioconcentration,

radiochemical purity, specific radioactivity, pH, residual solvents, and bacterial endotoxins

(LAL test). Standard operating procedures for these tests can be found in the quality

control and radiopharmacy forms.

8. Post-Synthesis Procedures:

8.1 HPLC and columns clean up: set the flow to 6 mL/min. and move the B pumps line into

the solvent bottle, which contains acetonitrile only. Keep the acetonitrile as 70%. Run

50-60 min and then stop running. Close the Beckman HPLC software.

8.2 Machine turn off: Turn off nitrogen and compressed air valves located outside hot-cell.

On the TRACERLab click synthesis – Reset in main menu to set all valves and vacuum on default status, then close the software. It is ready for next Box Clean procedure (SOP # MP201).

Synthesis comments:

_______________________________________________________________________________

___________________________________________________________________






Diagram 1. Layout of GE TRACERlab FXFN Module




SOP # QA301Standard Curve of Reference Fallypride



A) Stock solution of reference fallypride.

Weigh an accurate mass of fallypride (1−2 mg). Disolve in HPLC mobile phase (10−25

mL) in a volumetric flask for QC. Mix thoroughly for several minutes. When not in use,

store at −20 ○C in the freezer.

Concentration of solution = ~ 0.1 µg/µL

B) "Daily" dilution.

From the stock solution make a dilution 1: 100:

Example: take 100 µL of Stock solution.diluted with solvent (acetonitrile: HPLC

water 65: 35 v/v) in a volumetric flask (10 mL size)

Concentration: ~ 1−2 ng/1.0 µL

nmoles of fallypride (M.W. = 364.45) in this dilution:

1 ng/364.45 = 2.74 x 10-3 nmoles in 1.0 µL

Mix well to ensure homogeneity. When not in use, store at 4 ºC under refrigeration.

C) Procedure for obtaining standard curve

1. Open the Beckman 32 karat software and select the instrument ”Analysis System”

to start HPLC system.


S Purpose: To set a standard curve for authentic fallypride for calculation of the mass of fallypride in a in an analysis sample.



2. Set the analytical HPLC System (Beckman Coulter) in working conditions and

keep flow for 30 min for column equilibration.

HPLC System conditions:

Column: Prodigy ODS (3) (100 μ; 4.6 x 250 mm; Phenomenex)

Mobile Phase: Acetonitrile-ammonium formate (5 mM) (50: 50 v/v)

Flow Rate: 1.0 mL/min.

UV Wavelength: 305 nm

3. Select the method ”FallyprideQ.C” in method box and check the instrument setup

on the “Instrument Setup” box, then close box.

4. Using the "daily" dilution make injections into the HPLC system varying the injected

volume, measuring the areas and ensuring that plate count and peak shape is

consistent.

5. Suggested volumes of "Daily" dilution: 10, 20, 60, 80, 100 µL.

6. Make two to four injections of each volume.

7. Perform Regression Analysis on the data, plotting area units versus µg or µmoles.

8. Using the external or internal standards, calculate the slope of calibration line (a),

Y-axis intercept of calibration line (b) and correlation coefficient (r).9. Following is linear calibration fit of external or internal standards:

The equation for calculating the uncorrected amount is:

Y = ax+b





SOP # QA 304Analysis of Organic Residues by Gas Chromatography

in [18F]Fallypride for Injection



1. Responsibility -

1.1 It is the responsibility of the Chief of PET Radiopharmaceutical Sciences to ensure

that personnel are trained in this procedure.

1.2 It is the responsibility of personnel to adhere to this approved SOP.

1.3 Procedure adheres to GMP/GLP guidelines.

2. Scope - Quality control testing of volatile organic solvents in NIMH produced PET

radiopharmaceuticals.

3. Reference Document(s) –

3.1 6850A GC User Information

3.2 Installation, operation and maintenance manual for hydrogen

generator (Model H2-90; Parker Balston). (Bulletin TI-H2-90C)

4. Safety Precautions

4.1 Radiation Safety – ALARA (As Low as Reasonably Acceptable)

4.2 Chemical laboratory safety

5. Materials and equipment

5.1 Agilent 6850 GC with flame ionization detector (FID)

5.2 Agilent 6850 series autosampler

5.3 J & W DBWAX column, (30 m (l) x 0.25 mm (id) x 0.25 μm (film thickness) (Alltech,

part # 122-7032)


Purpose: to analyze for volatile solvent residues in [18F]Fallypride for Injection



5.4 Acquisition and data processing software: GC Chem Station (version: Rev. A.09.03

[1417] )

5.5 Inlet liner: split inlet glass liner with glass wool packing (Agilent part number, 5183-

469119251-60540)

5.6 Parker Balston H2-90 Hydrogen Generator

5.7 High purity grade (99.995 %) compressed helium compressed (Roberts Oxygen,

cat.no. R 102 F3)

5.8 In -house air purified by Parker Balston Zero Air Generator, Model 75-83NA

5.9 In -house deionized water (18 mega ohm) purified by Millipore Milli-Q j.) Autosampler

glass vial ( Agilent part no. 5182-0864) k.) Autosamper conical glass insert (Agilent

part no. 5183-2085)

5.10 Pipetman 200 L pipet (NIH stock # 6640-02-032-1955)

5.11 386 ppm internal standard aqueous solution of propionitrile prepared via 1 in 10

dilution of a 3860 ppm solution (0.5 mL propionitrile diluted to 100 mL mark with

water).

6. GC system configuration.

6.1 Injection port: split sample injection split ratio of 20:1, 250 ˚C

6.2 Carrier gas: Helium; 2 mL/min

6.3 Column temperature gradient: column temperature is initially operated at 50˚C and

held at this temperature for 1 min, and then increased to 150 ˚C at a rate of 20˚C /min.

The temperature is held at 150˚C for 0.5 min and then increased to 220˚C at a rate of

50˚C/min. After 3 min at 220˚C the column temperature is returned to starting

temperature of 50˚C.

6.4 Detector: FID with hydrogen at 40 mL/min and air at 450 mL/min. Helium make up

flow: 45 mL/min. Detector at 250 ˚C.

6.5 Autosampler: 10 L syringe. Sample injection volume: 1 L.

6.6 Needle/Syringe wash: four times prior to injection of sample and two times after

injection.




7. Procedure before data acquisition. Ensure that the water level in the H2-90 hydrogen

generator is above the lower limit. Otherwise, pour in 18 mega ohm water into the

reservoir until the level is just below the upper limit of water reservoir. Check the hydrogen

pressure is about 28 p.s.i. Check helium pressure is ca. 60 p.s.i. on main cylinder gauge.

Make sure that the solvents A and B (in the autosampler tray) for rinsing injection syringe

needle are filled with deionized (18 mega ohm water). Via a 200 L Pipetman pipettor,

pipette 50 L of [18F]Fallypride for Injection (test sample) into a glass insert housed in a GC

autosampler vial. Discard the radioactive pipette tip in radioactive waste. Add 50 L of the

internal standard solution of propionitrile (386 ppm) to the test sample. Cap the

autosampler vial with septum. Tap the bottom of the autosampler vial to remove air

bubbles. Place autosampler vial in autosampler rack and note rack position. On the GC

Control panel, use up or down arrow buttons to verify that FID is lit and the background

signal is ca. 5.

8. Data acquisition of [18F]FALLYPRIDE test sample spiked with internal standard. Double

click “Instrument 1 online” on the desktop. Select Method and Run Control from drop

down men bar below the file menu bar. Select ISPRN.M from drop down menu box on the

right side of Method and Run Control Box. Go to sequence on the main menu and select

sequence parameter. Type in subdirectory for radiopharmaceutical that will be tested (i.e.

Fallypride). Select Sequence Table from Sequence drop down menu. Enter 1 for the

number of injections per sample and location of the injection. Enter the sample name, file

name, and injection volume (1.0 L). When the system shows a ready sign (above start

button), hit the start button. To view on line signal, go to view on the main menu bar and

select on line signal/signal window 1. After 3.5 min, the GC chromatogram and report can

be generated; this is performed by going to the desktop and opening “Instrument 1 offline”.

On the left side of the screen, scroll the drop down menu to “data analysis”. Under the file

menu, scroll to “load signal. “ Find the file name in the appropriate subdirectory on the C

drive (e.g. C:\. . . \data\Fallpride). After loading the signal, scroll down to “print preview . . .

report” that is found in the file menu. The chromatogram and report should appear on the

computer screen and this can be printed out be hitting the print button on the bottom of the

report page. Attach the chromatogram and report to the quality control form. Note that

[18F]Fallypride fro Injection may be released before completion of the residual solvent test

(see section




9. Post-run method. Remove all samples and label radioactive samples. Download the

method Default.M that is used to maintain the oven temperature at 150 ˚C when GC is idle.





SOP #QA305

Analytical HPLC QC MethodApproved by: _____________________________Initials________ Date: _____________


Procedure

1. Set the analytical HPLC system (Beckman Coulter) in working conditions and

maintain flow for 40 min for column equilibration.

HPLC system conditions:

Column: Prodigy ODS (3) (100 μm; 4.6 x 250 mm; Cat. # 00G-4244-E0;

Phenomenex)

Mobile phase: Acetonitrile-5 mM ammonium formate (50: 50 v/v)

Flow rate: 1.0 mL/min.

UV Wavelength: 305 nm

2. Download the analytical method “Fally_analysis_secondSys” or “Fally_analysis_firstSys”

from method box in Beckman software.

3. Validation of HPLC system

Inject standard fallypride (20−40 µg), which is already prepared according to SOP

# QA301, and record the exactly mass (ng) injected and retention time of fallypride

standard.

Calculate the mass of fallypride corresponding to the peak of fallypride in HPLC

chromatography, based on the standard curve of fallypride.

Compare the mass of the fallypride injected with the mass calculated, the

difference should be equal or less then 10%. (Recovery ≤ 90%)

4. HPLC system cleaning

Inject solvent (100 µL) once or twice to clean the system in 10-15 min after

standard fallypride running. Check that only the solvent front peak should shows in

the chromatography.

Inject 10% ethanol in saline (100 µL). Check that only the solvent front and saline

peaks show in the chromatography.


Purpose: to perform analytical HPLC QC on [18F]Fallypride for Injection



5. QC of final product: [18F]Fallypride for Injection

Measure the radioactivity of sample of final product of [18F]Fallypride for Injection

(50−150 L) before and after injection onto the HPLC system in hot-cell 5, and

record the number in QC record sheet. Calculate the activity injected (decay

correlation)

Inject the sample of [18F]Fallypride for Injection (50−150 L depending on the

radioactivity concentration of formula).

Check the retention time (Rt) of the standard and the [18F]fallypride for

radiochemical identity (the difference of Rt < I min)

Calculate the mass corresponding the UV area of the [18F]fallypride peak in the

chromatograph, based on the standard curve of fallypride.

Calculate the specific radioactivity according to the radioactivity injected and the

mass calculated at HPLC injection time.

Calculate the specific activity at the time of HPLC injection, EOS and EOB (decay

correction).

Calculate the mass (g) of whole formula based on the radioactivity of final

formula, which recorded on master batch Sheet Page 4, and the specific activity at

EOS.

Record the chemical purity, radiochemical purity, the concentration of the final

formula and the expired time.

6. Post-analysis HPLC system and column cleaning: to run the HPLC system for 40−50 min

with the solvent (acetonitrile-HPLC water; 70: 30 v/v) for cleaning the HPLC system and

the column. Turn off the HPLC system and software.


SOP # QA306




Sterility TestApproved by: _____________________________Initials________ Date: _____________


Notes.

1. Routine sterility tests will be run by the Clinical Microbiology laboratory of the Department of

Laboratory Medicine at NIH, through a FDA approved procedure.

2. The radiochemistry lab will provide a sample of each formulation (in its original filtration

vial), 24 h after its preparation or when no traces of radioactivity are detected anymore (ten

or more half life periods).

3. Before their transportation to the testing laboratory, the samples will be stored in the

refrigerator until decay.

4. A log book will be kept in the radiochemistry lab indicating the date the samples were

transported, their lot number and the date and results of the test (readings at 7 and 14 d

should be indicated).



Purpose: To test the sterility of [18F]Fallypride for Injection



SOP # QA307

Pyrogen Test (LAL Bacterial Endotoxin Test). Simplified Procedure



MaterialsLAL test kit vials (e.g., Endosafe Inc., Charleston, SC or Cape Cod Associates, Falmouth MA)

Two vials 0.125 EU/mL(one for test sample, one for negative test)

One positive vial (5 EU/mL)

Sterile saline solution (from the same vial used for the final formulation)

Pyrogen-free test tubes (if not provided with kit)

Micropippets and tips (50 µL, 100 µL, 500 µL)

Tuberculin syringes (0.5 mL, 1 mL)

Incubator, 37°C

LAL Test Procedure: See “RADIOPHARMACY TEST RESULTS” form

Simplified Test Procedure ( it may not be used as a procedure of LAL test.)

a) From the final formulation in saline, take 50 µLand add to the test sample tube. To the

same LAL tube add 150 µLof saline (for a total of 200 µL).

b) To the negative test tube add 200 µL saline only.

c) To the positive test tube add also 200 µL saline.

d) Incubate all vials at 37 °C for 1 h. Remove the test vials at 20 min and in one smooth

careful motion, invert each tube (without totally disturbing the gel) and note gel

formation.

e) Continue incubation of the vials for the full 60 min. Again in one smooth motion, invert

each vial and note gel formation.


Purpose: to test the sterility of [18F]Fallypride for Injection



f) The test sensitivity equals the concentration of the test vial multiplied by the dilution

factor. If the test sample does not gel, report the result as less than the test sensitivity.

If the test sample gels, report as greater than or equal to the test sensitivity.

Note. The results at 20 min will allow release of the formulation for injection and the 60 min

samples will confirm the results.

Chemist_____________________________Initials_________________Date______________




SOP #QA308

Analysis of the Purity of Fallypride and Tosyl-Fallypride by HPLC



The HPLC System used in this method:

HPLC pump: System Gold 126 Solvent Model (Beckman Coulter)

UV detector: System Gold 166 Detector (Beckman Coulter)

Sample Injector: Rheodyne with 200 µLloop

The HPLC conditions used in this method:

Column: Prodigy ODS (10 µm, 4.6 X 250 mm, 100 A)

Mobile Phase: Acetonitrile-5 mM ammonium formate (50: 50 v/v) at 2 mL/min

Flow rate: 1 mL/min.

PDA: 254 nm

Procedure

1. Weigh 1−2 mg of precursor (tosyl-fallypride) in vial (20 mL size)

2. Dissolved the sample in of acetonitrile (10−20 mL). The concentration of this solution is

~ 1 µg/µL.

3. Inject 10 µLof the solution of tosyl-fallypride into HPLC and run for 30 min.

4. Use the Beckman method “Fallypride_Analysis_SecondSys” to interrogate the

Chromatograph, and get the Tosyl-fallypride purity (UV area percentage).

Chemist_____________________________Initials_________________Date__________


Purpose: to determine the purity of Tosyl-Fallypride by HPLC



AttachmentThis attachment lists the materials and equipments, which are used in the SOPs on Page 1.

Materials:

Item Name Name of supplierQuality grade and catalog #

1 4,7,13,16,21,24-Hexaoxa-1, 10-diazabicyclo

[8.8.8] hexacosane (Kryptofix 222)

Aldrich Chem. Co.

1001 West St. Paul

Ave., Milwaukee, WI

53233

Anhydrous, 98%

Cat. # 29110-1G

2 Potassium carbonate Aldrich Chem. Co.

1001 West St. Paul

Ave., Milwaukee, WI

53233

99.995%

Cat. # 367877-10G

3 Acetonitrile (anhydrous) Aldrich Chem. Co.

1001 West St. Paul

Ave., Milwaukee, WI

53233

Anhydrous, 99.8%

Cat. # 271004-100

ML

4 Acetonitrile Burdick and Jackson,

B&J Brand

HPLC Grade, ACS

Cat. # 017-4

5 Water Burdick and Jackson,

B&J Brand

HPLC Grade, ACS

Cat. # 4218

6 Acetone Fisher Scientific HPLC Grade, ACS

Cat #: 4218

7 Triethylamine Aldrich Chem. Co.

1001 West St. Paul

Ave., Milwaukee, WI

53233

HPLC Grade, ACS

Cat #: 471283-

500ML

8 Sodium acetate Aldrich Chem. Co.

1001 West St. Paul

Ave., Milwaukee, WI

53233

99+% ACS

Cat. #: 241245-

500G




9 Acetic acid Malinckodt

Chemicals

99+% ACS

Cat. # V193-14

10 Luna C18 (10 m; 100 Å; 250 x 21.2 mm) Phenomenex

2320 W. 205th St.

Torrance, CA 90501-

1456

Cat. # 00G-4094-

P0

11 Fallypride ABX,

Wilhelm-RÖnsch Str.

9. D-01454

Radeberg, Germany

> 90%

Cat. # 156.0025

12 Tosyl fallypride ABX,

Wilhelm-RÖnsch Str.

9. D- 01454

Radeberg, Germany

> 90%

Cat. # 155.0002

13 Ethyl alcohol (200 proof) The Warner-Graham

Company,

Cockeysville, MD

CAS # 64-17-5

14 Sodium Chloride for Injection (0.9% w/v;

USP

American

Pharmaceutical

Partner, Inc.

USP

CAT#:918610

15 Sterile empty vial (10 mL) Abbott laboratories USP

Cat. # 5816-11

16 Sep-pak Cartridges Plus C18 Waters Corporate

34 Maple Street,

Milford,

MA. 01757 USA

Cat. # WAT020515

17 Millex filter (25 mm; 0.22 m pore size) Millipore Cat. # SLGVR35LS

18 Millex GV filter (25 mm; 0.22 m pore size;

4 mm diameter)

Millipore Cat. # SLGV004SL




Instruments and equipment:

Operation/Function Manufacturer Model Serial #

Radiosynthesis General Electric TRACERlab FXFN 14390

HPLC purification Beckman Coulter System Gold 126

Solvent Model

342-2187

UV absorbance detection

in HPLC purification

Beckman Coulter System Gold 166

Detector

332-2187

Radioactivity detection in

HPLC purification

Bioscan Flow Counter 0409-316


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